Isolated DNA molecule encoding SHET1 of Shigella flexneri 2a and mutant Shigella flexneri 2a

ABSTRACT

Substantially pure enterotoxins of Shigella flexneri 2a are described, along with a method for obtaining the same, antibodies having binding specificity to the enterotoxins and a method for use of the enterotoxins to develop a non-reactogenic Shigella flexneri 2a vaccine candidate.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a Continuation-in-part of U.S. patent application Ser. No.08/160,317, filed Dec. 2, 1993, now U.S. Pat. No. 5,468,639, which inturn is a Continuation-in-part of U.S. patent application Ser. No.07/894,774, filed Jun. 5, 1992, now abandoned.

FIELD OF THE INVENTION

The present invention relates to two substantially pure enterotoxins ofShigella flexneri 2a (hereinafter "SheT1" and "ShET2"), a method forobtaining the same, antibodies having binding specificity to theenterotoxins and a method for use of the enterotoxins to develop anon-reactogenic Shigella flexneri 2a vaccine candidate.

BACKGROUND OF THE INVENTION

Much has been written about the molecular pathogenesis of Shigella withrespect to the genes and gene products involved in their ability toinvade epithelial cells, and thereby to cause dysentery (Makino et al,Microb. Pathog., 5:267-274 (1988); Sansonetti et al, Infect. Immun.,35:852-860 (1982); Hale et al, Infect. Immun., 40:340-350 (1983); Pal etal, J. Clin. Microbiol., 27:561-563 (1989); and Venkatesan et al, Proc.Nat'l. Acad. Sci. U.S.A., 85:9317-9321 (1988)). In contrast,surprisingly little is known of the precise mechanisms by which Shigellacause watery diarrhea.

Although the cardinal feature of the pathogenesis of Shigella flexneri2a infection involves the invasion of epithelial cells, because Shigellaflexneri 2a can cause watery diarrhea, it has been hypothesized thatShigella flexneri 2a also produces an enterotoxin (Rout et al,Gastroenterology, 68:270-278 (1975); and Kinsey et al, Infect. Immun.,14:368-371 (1976)). More specifically, the following observations havesuggested the existence of enterotoxins in Shigella flexneri 2a:

1. Clinically in humans Shigella flexneri 2a infections are usuallycharacterized by a period of watery diarrhea that precedes the onset ofscanty dysenteric stools of blood and mucus (DuPont et al, J. Infect.Dis., 119:296-299 (1969); and Stoll et al, J. Infect. Dis., 146:177-183(1982)). In mild cases, only watery diarrhea may occur, leading to aclinical picture undistinguishable from that due to enterotoxingenic E.coli infection (Taylor et al, J. Infect. Dis., 153:1132-1138 (1986); andTaylor et al, J. Clin. Microbiol., 26:1362-1366 (1988)).

2. When Shigella are fed to monkeys, three clinical syndromes are seen(Route et al, Gastroenterology, 68:270-278 (1975)). Some monkeys developonly dysentery; some exhibit only watery diarrhea and some exhibitwatery diarrhea and dysentery. In vivo perfusion studies by Rout et al,Gastroenterology, 68:270-278 (1975)) showed that net transport of waterinto the lumen of the colon occurs in all ill animals. In contrast, onlyin the jejunum of monkeys with overt watery diarrhea (alone or followedby dysentery) does there occur net secretion of water, sodium andchloride ions; such net transport does not occur in the jejunum ofmonkeys manifesting dysentery without watery diarrhea. Net secretion inthe jejunum was not accompanied by abnormal histological findings inthis anatomic site of the small intestine.

3. The net secretion of water and electrolytes into the jejunum ofmonkeys with watery diarrhea requires the passage of Shigella throughthe jejunum (Kinsey et al, Infect. Immun., 14:368-371 (1976)). This wasdemonstrated by bypassing the small intestine and inoculating Shigelladirectly into the cecum of monkeys. Of 16 monkeys who developed clinicalillness, manifested dysentery, ". . . only rarely preceded by milddiarrhea". Net secretion of water and sodium into the colon was recordedin ill monkeys that developed dysentery following intracecalinoculation, while no abnormalities of water or electrolyte transportwere observed in the jejunum of the ill animals.

Together, these observations suggest that Shigella elaborate anenterotoxin that elicits secretion early in the infection as theorganisms pass through the jejunum.

However, except for the cytotoxin/neurotoxin/enterotoxin elaborated byShigella dysenteriae (O'Brien et al, Microbiol. Rev., 51:206-220 (1987);Keusch et al, Pharmac. Ther., 15:403-438 (1982); and Fontaine et al,Infect. Immun., 56:3099-3109 (1988)), but not by other Shigella species,little convincing proof has been generated to substantiate thecontention that Shigella, other than Shigella dysenteriae, in factproduce enterotoxins.

More specifically, previous attempts in the art to detect enterotoxicactivity in supernatants of Shigella flexneri 2a have yielded positivefindings in only one instance. O'Brien et al, Infect. Immun., 15:796-798(1977), partially purified a toxin produced by Shigella flexneri 2astrain M4243 that was detectable in cell-free supernatants. This toxinstimulated fluid production in rabbit ileal loops, but was alsocytotoxic for HeLa cells in monolayers and was lethal when inoculatedintraperitoneally into mice. Further, it was not necessary to grow thebacteria in Fe⁺⁺ -depleted medium in order to detect the enterotoxicactivity. In addition, the cytotoxicity of the toxin described byO'Brien et al, supra, was neutralized by anti-sera to Shiga (Shigelladysenteriae 1) toxin.

Enterotoxic activity in cell-free supernatants of Shigella flexneri 2aand 3a was reported by Ketyi et al, Acta Microbiol. Acad. Sci. Hung.,25:165-171 (1978); Ketyi et al, Acta Microbiol. Acad. Sci. Hung.,25:219-227 (1978); and Ketyi et al, Acta Microbiol. Acad. Sci. Hung.,25:319-325 (1978). Filtered ultrasonic lysates of two Shigella flexneri2a and 3a strains were founds to give rapid fluid accumulation in rabbitileal loops (4 hour assay). However, the loops showed no fluidaccumulation when examined at 18-24 hours after inoculation. Only threeloops were inoculated for each of the two test strains and when examinedat 4 hours, only 2/3 for one strain and 1/3 for the other strain werepositive. In addition, the Shigella were not cultured in Fe⁺⁺ -depletedmedium.

In the present invention, it was discovered for the first time thatenterotoxic activity, which is clearly dissociated from cytotoxicactivity, is expressed by Shigella flexneri 2a in the bacteria-freeculture supernatant, and could be detected only after growth of thebacteria in Fe⁺⁺ -depleted medium.

It has been reported that when grown in Fe⁺⁺ -depleted medium,enteroinvasive Escherichia coli (EIEC) elaborate an enterotoxin (MWcirca 68-80 kDa) that causes fluid accumulation in isolated rabbit ilealloops and an electrical response in Ussing chambers (Fasano et al,Infect. Immun., 58:3717-3723 (1990)). Based on the similarities known toexist between enteroinvasive E. coli and Shigella (Levine et al, J.Infect. Dis., 155:377-389 (1987)), it was postulated in the presentinvention that Shigella flexneri 2a would express an enterotoxin whengrown in Fe⁺⁺ -depleted medium.

In the present invention, it was unexpectedly disclosed that Shigellaflexneri 2a produces two distinct enterotoxins, one encoded by thechromosome, and the other encoded by an invasiveness virulent plasmid.The latter enterotoxin was found in the present invention to beessentially the same as the EIEC enterotoxin.

SUMMARY OF THE INVENTION

An object of the present invention is to purify the two enterotoxinsproduced by Shigella flexneri 2a.

Another object of the present invention is to provide a method forculturing Shigella flexneri 2a so as to produce said enterotoxins.

A further object of the present invention is to provide antibodieshaving binding specificity for said enterotoxins.

An additional object is to identify, clone and sequence the genesencoding such enterotoxins.

Still another object of the present invention is provide Shigellaflexneri 2a mutants which fail to produce at least one functionalenterotoxin as a result of a mutation in a Shigella enterotoxin gene.

These and other objects of the present invention have been achieved inthe detailed description of the invention provided below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of assays for enterotoxic activity in Ussingchambers when using culture supernatants of Shigella flexneri 2a strainsM4243, M4243avir and BS103, the 30-100 kDa fraction of EIEC strainCVD/EI-34 (0136:H-) (as a positive control) and culture media (as anegative control). In these assays, variations in short-circuit current(delta I_(sc)) were measured.

FIGS. 2A-2D show the results of assays for enterotoxic activity inUssing chambers when using Shigella flexneri 2a strain M4243 culturesupernatant which was first neutralized with anti-sera against theShigella flexneri 2a enterotoxins (anti-shETs), with anti-sera againstthe EIEC enterotoxin (anti-EIET), with pre-challenge sera orpost-challenge sera of volunteers challenged with wild-type Shigellaflexneri 2a. In these assays, variations in short-circuit current (deltaI_(sc)) (FIG. 2A and 2C and transepithelial electrical potentialdifferences (delta PD) (FIG. 2B and 2D) were measured.

FIGS. 3A-3B shows the molecular mass determination of the Shigellaflexneri 2a strain M4243 enterotoxic moieties when assayed in rabbitileal loops (FIG. 3A) and in Ussing chambers (FIG. 3B). In the rabbitileal loop assays, fluid accumulations were measured and in the Ussingchambers, variations in short-circuit current (delta I_(sc)) weremeasured.

FIG. 4 shows the results of assays for enterotoxic activity in Ussingchambers when using protein bands from SDS-PAGE obtained from strainM4243avir (containing only ShET1 enterotoxin) that represent the 65-75kDa column fraction, an extract of an unused strip of nitrocellulose(negative control), and a sample representing the 65-75 kDa columnfraction (positive control). Values are given as variation (μAmp/cm²)with N representing the number of observations on independently preparedsamples.

FIG. 5 shows a restriction map of the fragments in pJS26 which containsthe tie gene, as well as restriction maps for the relevant portions ofplasmids derived from pJS26.

FIGS. 6A-6D (SEQ ID NO:1) show the DNA sequence of EIET enterotoxinencoded by enteroinvasive E. coli, as well as the determined amino acidsequence.

FIGS. 7A-7D (SEQ ID NO:2) show the DNA sequence of ShET2 enterotoxinlocated on the Shigella flexneri 2a invasiveness plasmid, as well as thedetermined amino acid sequence.

FIG. 8 shows the restriction map of the fragment in pF9-1-90 whichcontains the ShET1 gene.

FIGS. 9A-9B (SEQ ID NO:15) show the DNA sequence of ShET1 enterotoxinlocated on the Shigella flexneri 2a chromosome, as well as thedetermined amino acid sequence.

DETAILED DESCRIPTION OF THE INVENTION

In the present invention, the enterotoxins are obtained by culturingShigella flexneri 2a in Fe⁺⁺ -depleted medium and collecting thesupernatant.

"Fe⁺⁺ -depleted media" is an expression well-known and used in the art.This expression refers to iron-depleted media, such as syncase broth,treated, e.g., in CHELEX® (BioRad), a styrene divinyl benzene resinmatrix with iminodiacetic acid exchange groups, to leave just traces ofiron in the medium.

The particular culture medium employed is not critical to the presentinvention. Examples of such culture media include Fe⁺⁺ -depleted syncasebroth or L-broth plus ethylenediamine-N-N'-diacetic acid (EDDA). Fe⁺⁺-depleted syncase broth is the preferred culture medium since maximalproduction of the enterotoxin was obtained with this medium.

While the culture temperature and incubation period are not critical tothe present invention, generally the culturing temperature will rangefrom 30° to 37° C., preferably 36° to 37° C., and the incubation periodwill range from 24 to 72 hours, preferably 48 to 72 hours.

The enterotoxins can be purified from the supernatant by size exclusionand HPLC chromatography.

Shigella flexneri 2a is a well-known virulent Shigella serotypeavailable from a variety of sources, such as the Center for VaccineDevelopment, the Center for Disease Control, the Walter Reed ArmyInstitute of Research, the Uniformed Services University of the HealthSciences, and the Institut Pasteur. The particular strain of Shigellaflexneri 2a employed in the present invention is not critical thereto.Examples of such Shigella flexneri 2a strains include M4243, M4243avir,Shigella flexneri 2a Chile 747, Shigella flexneri 2a Chile 3480(Ferreccio et al, Am. J. Epi., 134:614-627 (1991)); strain 2457T(Kotloff et al, Infect. Immun., 60:2218-2224 (1992); and BS103 (Andrewset al, Infect., Immun., 59:1997-2005 (1991)). The preferred Shigellaflexneri 2a strains employed in the present invention are Shigellaflexneri 2a strain M4243 and M4243avir.

Shigella flexneri 2a strain M4243 and its plasmid-cured derivativeM4243avir can be obtained from, e.g., Dr. Samuel B. Formal of the WalterReed Army Institute of Research, Washington, D.C. BS103 can be obtainedfrom Dr. Anthony Maurelli of the Uniformed Services University of theHealth Sciences, Bethesda, Md.

The antibodies having binding specificity to the two enterotoxins of thepresent invention may be polyclonal or monoclonal. Polyclonal antibodiesto the purified enterotoxins can be prepared by conventional means asdescribed in Antibodies: A Laboratory Manual, Harlow and David Lane,Eds., Cold Spring Harbor Laboratory Press (1988). Monoclonal antibodiesto the purified enterotoxins can be prepared by conventional means asdescribed in Kohler et al, Nature, 256:495-497 (1975).

Monoclonal antibodies obtained using purified enterotoxins may be usedto induce a passive immunity against Shigella enteric infection. Suchantibodies will bind Shigella flexneri 2a enterotoxins, thus preventingthese interaction with the cellular receptor, and preventing thestimulation of water and electrolyte secretion. The total amount ofantibodies used to induce passive immunity is generally about 10 mg to10 g. The total amount of toxoid used to produce such antibodies isgenerally about 500 μg to 5.0 mg.

The substantially pure enterotoxins of the present invention are alsouseful for the development of a non-reactogenic Shigella flexneri 2acandidate live oral vaccine. As background, in the United States,Shigella flexneri 2a is one of the most common serotype of Shigellaassociated with disease. In developing countries of the world, Shigellaflexneri is the most common serogroup of Shigella causing diarrhealdisease and Shigella flexneri 2a is often the single most commonserotype. Prospective epidemiologic studies in a low socioeconomiccommunity in Santiago, Chile, where Shigella infections are endemic,have shown that an initial clinical episode of shigellosis conferssignificant protection against subsequent disease due to the sameserotype (Ferroccio et al, Am. J. Epidemiol., 134:614-627 (1991)). Theimmunizing effect of diarrheal illness due to wild-type Shigella hasalso been demonstrated in a volunteer model of experimental shigellosiswhere an initial clinical infection due to Shigella flexneri 2a (DuPontet al, J. Infect. Dis., 125:12-16 (1972)) or Shigella sonnel (Herringtonet al, Vaccine, 8:353-357 (1990)) conferred significant protectionagainst re-challenge with the homologous wild-type organism. Togetherthese observations suggest that it may be possible to protect againstshigellosis with a vaccine that requires only a single dose.

There have been many attempts to develop attenuated strains of Shigellato serve as vaccines. Some attempts have met with limited success. Inthe 1960s, streptomycin-dependent strains of Shigella flexneri 2a andother serotypes were developed and utilized as live oral vaccines (Melet al, Bull. WHO, 32:647-655 (1965); Mel et al, Bull. WHO, 39:375-380(1968); and Mel et al, Acta Microbiol. Acad. Scient. Hung., 21:109-114(1974)). These streptomycin-dependent strains were safe and conferredsignificant serotype-specific protection against shigellosis in most ofthe controlled field trials of efficacy that were carried out (Mel etal, Bull. WHO, 32:647-655 (1965); Mel et al, Bull. WHO, 39:375-380(1968); Mel et al, Acta Microbiol. Acad. Scient. Hung., 21:109-114(1974); and Levine et al, Am. J. Epidemiol., 133:424-429 (1976)).However, the streptomycin-dependent Shigella vaccinees suffer fromcertain drawbacks. One is the fact that multiple spaced doses have to begiven to confer protection (four doses over a two-week period containinglarge numbers (2-4×10¹⁰) of viable vaccine organisms). Moreover,protection is relatively short-lived. A booster dose has to be givenafter one year in order to maintain protection (Mel et al, ActaMicrobiol. Acad. Scient. Hung., 21:109-114 (1974)). Colonial mutantShigella flexneri 2a vaccine strain T₃₂ described in Istrari et al,Arch. Roumaines Pathol. Exp. Microbiol., 24:677-686 (1985), is alsowell-tolerated and protective (Wang Bing Rui, Arch. Roumaines Pathol.Exp. Microbiol., 43:285-289 (1984)), but still requires multiple doses.

Because of the above-mentioned drawbacks of the streptomycin-dependentand T₃₂ vaccines of the 1960s, various investigators have attempted tomake more immunogenic Shigella vaccines that can protect following theadministration of just a single dose. The approaches taken haveincluded:

(1) introducing specific segments of the chromosome of E. coli K-12 intoShigella by conjugation (Formal et al, Dev. Biol. Stand., 15:73-78(1971); and Levine et al, J. Infect. Dis., 127:261-270 (1973));

(2) introducing DNA encoding protective Shigella antigens into E. coliK-12 (Formal et al, Infect. Immun., 46:465-469 (1984)); and

(3) inactivating genes of the aromatic amino acid biosynthesis pathway,thereby rendering the Shigella nutritionally dependent on substratesthat are not available in human tissues (Lindberg et al, Vaccine,6:146-150 (1988); and Karnell et al, Rev. Infect. Dis., 13(4):S357-361(1991)).

Regrettably, each of the above approaches has met with limitations. Thatis, hybrids in which Shigella carrying attenuating E. coli DNA areunstable and can revert to full virulence (Levine et al, J. Infect.Dis., 127:261-270 (1973)). Further, the most recent generation of E.coli expressing Shigella antigens has been associated with sidereactions in vaccinees, including fever, mild diarrhea and everydysentery in some individuals (Kotloff et al, Infect. Immun.,60:2218-2224 (1992)). Finally, some recipients of ΔaroD Shigellaflexneri developed mild diarrhea (Karnell et al, Rev. Infect Dis.,13(4):S357-361 (1991)). It has been hypothesized in the presentapplication that the residual diarrhea encountered in these variousShigella flexneri candidate vaccine strains is likely due to the twoenterotoxins.

Accordingly, Shigella flexneri 2a vaccine candidates can be constructedwhich, e.g., in addition to containing other attenuating mutations,express one or two toxoids, rather than the enterotoxins. This can beaccomplished by deleting the portion of the enterotoxin genes thatencodes the biologically active "toxic" site, leaving intact immunogenicsequences of the protein. Specifically, a Shigella flexneri 2a strain inwhich deletion mutations are introduced in at least one aro gene (aroA,aroC, or aroD) of the Shigella chromosome, rendering the strainauxotrophic for paraaminobenzoic acid, a substrate that cannot besufficiently scavenged in vivo in humans, can be constructed, such asstrain CVD1203 (ATCC No. 55556) prepared in Example 8 below.

In addition, the strain will preferably have an independentlyattenuating, deletion mutation in the virG gene, which is found on the140 MD invasiveness plasmid of Shigella flexneri 2a. This plasmid gene,also known as icsa (Sansonetti et al, Vaccine, 7:443-450 (1989)), isinvolved with the intracellular and intercellular spread of Shigella.This mutation is also present in CVD1203.

Recognizing that the vaccine candidate, e.g., CVD1203, may still not besufficiently attenuated with just these mutations (since the ability toproduce enterotoxins remains intact), the enterotoxin genes can bemutated. One type of mutation, e.g., a deletion of substantially all ofthe enterotoxin genes, will totally inactivate enterotoxin production,resulting in a non-enterotoxinogenic strain. A second mutation, e.g., adeletion of part of the enterotoxin genes, will result in expression oftoxoids, i.e., modified proteins that lacks the toxicity of the toxinsbut retains immunogenic moieties. This alternative mutation will resultin a vaccine candidate strain that expresses two toxoids. These toxoidscan be used to induce active immunity against Shigella flexneriinfection.

The particular size of the deletion is not critical to the presentinvention, and can be readily determined based upon whether one desiresto totally inactivate the enterotoxins, or simply produce toxoids. Asshown in Example 7, ShET1 is encoded by two distinct genes (FIGS. 9A and9B, Seq. ID NO:15). Based on similarities between ShET1 genes and genesencoding for other endotoxins, such as cholera toxin or heat-labileenterotoxin of enterotoxigenic E. coli, the large orf encodes for theactive subunit. Thus, an internal deletion of this orf should give riseto the production of an immunogenic toxoid.

The isolated DNA molecules of the present invention encoding theenterotoxin genes can be cloned in any suitable plasmid or vector, andused, e.g., to produce large amounts of DNA for use as probes or tointegrate mutated enterotoxin genes into vaccine strains.

The expression "isolated" is used herein to mean set apart from itsnatural environment, e.g., the DNA molecules are separated from theparent chromosome or parent plasmid from which they were originallyobtained in the present invention. Thus, "isolated" as used hereinincludes the presence of the DNA molecules in a foreign host or foreignplasmid.

The following examples are provided for illustrative purposes only andare in no way intended to limit the scope of the present invention.

EXAMPLE 1 Production of Enterotoxins

A. Preparation of Culture Filtrate Fraction

Shigella flexneri 2a strain M4243 and its plasmid-cured derivativesM4243avir and BS103, were grown overnight at 37° C. with shaking (200rpm) in 5.0 ml of CHELEX® (BioRad, Richmond, Calif.) treated, Fe⁺⁺-depleted syncase broth (O'Brien et al, J. Infect. Dis., 136:763-759(1982)). CHELEX® binds to the iron present in the broth. All culturevessels employed were either new plastic or borosilicate glass soakedovernight in 6.0N HCl, and rinsed in distilled deionized water to ensurethe absence of iron. 50 μl of the resulting culture broth were thensubcultured in 5.0 ml of Fe⁺⁺ -depleted syncase broth in baffledFernbach flasks, and incubated for an additional 48 hours under theabove conditions. After 72 hours of incubation, the cultures wereharvested by centrifugation of 12,000×g for 20 minutes at 4° C. and thesupernatants were passed through a 0.45 μm filter membrane (MilliporeProducts, Bedford, Mass.) to obtain a "sterile supernatant".

B. Rabbit Ileal Loop Test

Whole cultures of Shigella flexneri 2a strain M4243 and itsplasmid-cured derivative M4243avir, along with their respective sterilesupernatants, obtained as described above, were tested in a standardrabbit ileal loop test. Supernatants of EIEC strain CVD/EI-34 (0136:H-)(which induces fluid accumulation in rabbit ileal loops) andnon-pathogenic E. coli HS, were also included in each experiment aspositive and negative controls, respectively (Fasano et al, Infect.Immun., 58:3717-3723 (1991)). EIEC strain CVD/EI-34 (0136:H-) wasobtained from the Center for Vaccine Development strain collection. E.coli HS was obtained from Dr. Herman Schneider, Walter Reed ArmyInstitute of Research.

More specifically, male adult New Zealand white rabbits weighing 2-3 kgwere starved for 24 hours but allowed water ad libitum. These animalswere then anesthetized by intramuscular administration of a cocktail of50 mg/kg ketamine and 1.0 mg/kg acepromazine, followed by intramuscularadministration of 7.0 mg/kg xylazine.

Bacterial cultures were grown to reach 10⁸ -10⁹ CFU/ml. Whole cultures,or the respective sterile supernatants, in a standard volume of 1.0 ml,were injected into the lumen of the intestine of the anesthetizedrabbits near a tie closest to the mesoappendix (Moon et al, Ann. NY.Acad. Sci., 176:197-211 (1971)); a second tie was made to isolate thesite of inoculation. Proceeding proximally along the ileum, a series offive to six loops 7-8 cm long separated by double ties were isolated andinoculated (Moon et al, Ann. NY. Acad. Sci., 176:197-211 (1971)). After18 hours of incubation, the animals were sacrificed, the fluid volumeand length of the loops were measured, and sections of intestine fromeach loop were fixed in 10% (v/v) formalinized saline and examined bylight microscopy. The results of the loop test are shown in Experiment 1in Table 1 below.

                  TABLE 1                                                         ______________________________________                                        Fluid Accumulation (ml/cm) in Rabbit Ileal Loops                              ______________________________________                                        Experiment 1                                                                  M4243 bacteria (5)    1.06 ± 0.34*                                         M4243 supernatant (5)                                                                               0.52 ± 0.10**                                        M4243avir bacteria (5)                                                                             0.21 ± 0.50                                           M4243avir supernatant (5)                                                                          0.24 ± 0.09                                           HS supernatant (5)   0.09 ± 0.06                                           Experiment 2                                                                  M4243 supernatants:                                                           L broth, 24 hours (4)                                                                              0.01 ± 0.01                                           L broth, 72 hours (4)                                                                              0.04 ± 0.03                                           Minimal Fe.sup.++  broth, 24 hours (4)                                                              0.43 ± 0.11*                                         Minimal Fe.sup.++  broth, 72 hours (4)                                                              0.47 ± 0.14*                                         HS supernatant:                                                               Minimal Fe.sup.++  broth, 24 hours (4)                                                             0.01 ± 0.01                                           ______________________________________                                         In the Table above, the results are expressed as mean ± SE for (n)         animals. The bacterial cultures were grown for 72 hours unless otherwise      indicated.                                                                    *p < 0.01 compared to HS; ** p < 0.05 compared to HS.                    

As shown in Experiment 1 in Table 1 above, the intestinal loops injectedwith the positive control, i.e., whole viable cultures of M4243, andsterile culture supernatant therefrom, showed pronounced fluidaccumulation at 18 hours post-inoculation, with the whole viable cultureshowing a two-fold greater fluid accumulation. Further, as shown inExperiment 1 in Table 1 above, fluid accumulation induced by M4243avir(both whole culture and sterile supernatant) was not significantlyhigher than the negative control strain HS.

The fluid to gut length recorded in the rabbit ileal loops, 0.5 ml/cm,measured using graduated syringes (fluid) and a scale (length), wassubstantially less than seen with enterohemorrhagic E. coli (EHEC)strain 933J, serotype (0157:H7), where ratios of 1.5-2.0 ml/cm occur.However, the recorded fluid to gut length measured using graduatessyringes (fluid) and a scale (length) still represents definite evidenceof net secretion and fluid accumulation.

On histologic examination of the sections of intestine from each loop,severe tissue damage was observed with whole cultures of M4243,characterized by prominent necrosis of the luminal epithelium and markedvillus atrophy. In contrast, with M4243 sterile culture supernatant, notissue damage was detected. Further, no tissue damage was observed withwhole cultures of M4243avir or sterile supernatants therefrom. Moreover,no tissue damage was observed with tissue incubated with the negativecontrol strain HS.

To determine whether the time of incubation and the iron content in themedium are crucial for the full expression of this enterotoxic moiety,Shigella flexneri 2a strain M4243 was cultured in Fe⁺⁺ -containingmedium (L-broth) and Fe⁺⁺ -depleted medium (syncase broth). After 24 and72 hours of incubation for each medium, sterile, supernatants wereobtained and then rejected in ileal loops, as described above. Theresults are shown in Experiment 2 in Table 1 above.

As shown in Experiment 2 in Table 1 above, Fe⁺⁺ -depleted cultureconditions are required in order to detect expression of theenterotoxin. Further, enterotoxin expression was not notably affected bythe length of incubation.

The results obtained in the rabbit ileal loop assay were compatible withelaboration of an enterotoxin by M4243.

C. Ussing Chambers

These experiments were performed as previously described by Guandaliniet al, J. Pediatr. Gastroenterol. Nutr., 6:953-960 (1987). Briefly, maleadult New Zealand white rabbits weighing 2-3 kg were anesthetized bymethoxyflurane inhalation and then sacrificed by air embolism. A 20 cmsegment of distal ileum was removed, opened along the mesenteric border,rinsed free of intestinal contents, and stripped of muscular and serosallayers. Four pieces of intestine so prepared were then mounted in luciteUssing chambers (1.12 cm² opening) and bathed in Ringer's solutioncontaining 53 mM NaCl, 5.0 mM KCl, 30.5 mM Na₂ SO₄, 30.5 mM mannitol,1.69 mM Na₂ HPO₄, 0.3 mM NaH₂ PO₄, 1.25 mM CaCl₂, 1.1 mM MgCl₂ and 25 mMNaHCO₃. During the experiment, the tissue was kept at 37° C. and gassedwith 95% 0₂ -5% CO₂. Once the tissue reached a steady-state condition,300 μl of either M4243, M4243avir or BS103 sterile supernatants fromFe⁺⁺ -depleted cultures were added to the mucosal surface, resulting ina 1:33 dilution of the original culture filtrate concentration (0.3 mlinto 10 ml of Ringer's solution). 300 μl of either M4243, M4243avir orBS103 sterile supernatants were also added to the serosal side topreserve osmotic balance. Variation in transepithelial electricalpotential difference (delta PD), total tissue conductance (Gt) andshort-circuit current (delta I_(sc)) were recorded. The 30-100 kDasupernatant fraction from EIEC (0136:H-) and CHELEX®-treated syncasebroth (culture media) were also tested in the same manner as positiveand negative controls, respectively. Four animals were employed for eachtest. The results are shown in FIG. 1.

As shown in FIG. 1, the overall increase in I_(sc) was significantlygreater for the M4243 supernatant as compared to the negative control(culture medium) (**=p<0.02), and similar in magnitude to that inducedby the positive control (EIEC 0136:H-). On the other hand, supernatantfrom the plasmid-cured derivatives M4243avir and BS103 expressedsignificantly less enterotoxin in comparison with the plasmid-containingparent strain (*=p<0.05). However, the enterotoxic activity of theM4243avir and BS103 supernatants was nevertheless significantly greaterthan the negative control (culture medium) (*=p<0.05). Possibleinterpretations of such results include: (1) a plasmid-encodedregulation factor that regulates a chromosomal toxin gene; (2) multiplecopies of the same gene located both on the S. flexneri 2a chromosomeand the plasmid; or (3) a gene on the invasiveness plasmid encoding fora distinct enterotoxic factor. As discussed in detail below, this lasthypothesis turned to be correct.

The plasmid-cured derivative of strain M4243 showed less enterotoxicactivity compared to the wild-type in both ileal loops and in Ussingchambers. Only in Ussing chambers did M4243avir induce changes that weresignificantly different from the negative control; this could be due tothe higher sensitivity of the Ussing chamber technique as compared tothe ileal loop assay. These data suggest that, while not absolutelynecessary for the effect, the virulence plasmid of Shigella flexneri 2aM4243 enhances enterotoxic activity.

D. Enterotoxin Neutralization

EIEC (0136:H-) and Shigella flexneri 2a share many similarities, e.g.,surface antigens, identical plasmids (pInv), clinical manifestations,etc. Thus, neutralization experiments were carried out to determine ifthere is any immunological relatedness between the enterotoxin producedby EIET (0136:H-) and the enterotoxin produced by M4243.

More specifically, 600 μl of the 30-100 kDa fraction of M4243 sterilesupernatant (see Section E. below) were incubated for 60 min at 37° C.with 60 μl of anti-ShET polyclonal sera (anti-Shigella flexneri 2aenterotoxin) or with anti-EIET polyclonal sera (anti-enteroinvasive E.coli enterotoxin) or with pre- or post-challenged convalescent sera.

Anti-ShET polyclonal sera, anti-EIET polyclonal sera, and convalescentsera were obtained as described in Example 2.

The resulting samples were tested in Ussing chambers as described inSection C. above with half of each mixture added to each side of achamber. The results are shown in FIGS. 2A-2D.

As shown in FIGS. 2A-2D, the electrical response in Ussing chambers wasdrastically reduced when M4243 supernatant was pre-incubated withpolyclonal rabbit antibodies raised against the Shigella flexneri 2aenterotoxins (anti-ShETs) or with convalescent sera from volunteers whohad been challenged with Shigella flexneri 2a. This neutralization wasnot observed in either of the pre-immune sera control experiments inwhich responses were similar to those seen when testing the activefraction alone.

Only a partial cross-neutralization was observed when the M4243supernatant was pre-incubated with polyclonal antibodies raised againstthe enteroinvasive E. coli enterotoxin (anti-EIET).

In FIGS. 2A-2D, the number of animals tested was 4. Values are mean ±SE.*=p<0.05 and * *=p<0.02 compared to PBS (the negative control).

Taken together, these results suggest that S. flexneri supernatantprobably contains two enterotoxin moieties, ShET1 (whose gene is locatedon S. flexneri chromosome) and ShET2 (whose gene is located on theinvasiveness plasmid). Both enterotoxins were neutralized when anti-S.flexneri 2a antiserum was used. The ability of EIEC antiserum topartially neutralize the S. flexneri 2a supernatant enterotoxicity wasdue to the high similarity (99%) of EIET gene with ShET2 gene (seebelow).

E. Estimate of Molecular Mass

To obtain an estimate of the M_(r) of the Shigella flexneri 2aenterotoxins, sterile supernatant of M4243 was fractionated byultracentrifugation through DIAFLO ultrafiltration membranes (AmiconCorp., Danvers, Mass.). YM100 (100,000-MW cutoff) and YM30 (30,000-MWcutoff) membranes were utilized to produce fractions defined by thesesize limits. Membrane retentates were washed free of lower molecularweight species with phosphate buffered saline (pH 7.3) (PBS), by twosuccessive 10:1 volume dilutions with PBS, reconcentration, and finalreconstitution to the original volume in PBS.

The individual fractions, representing coarse molecular weight poolsof >100 kDa, 30-100 kDa and 0.5-30 kDa, were tested for enterotoxicactivity in Ussing chambers and ileal loops. The results are shown inFIG. 3A-3B.

As shown in FIGS. 3A-3B, both ileal loop (FIG. 3A) and Ussing chamber(FIG. 3B) assays localized the active enterotoxic fraction within the30-100 kDa size range.

In FIGS. 3A-3B, the number of animals tested was 4. Values are means±SE. *=p<0.05 and **=p<0.02 compared to the other fractions and thenegative control.

F. Cytotoxicity Assay

To establish whether there is a correlation between enterotoxic activityand cytotoxic activity, the following experiments were carried out.

A cell lysate was obtained as follows: Cultures from strain M4243 wereharvested by centrifugation at 12,000×g for 20 minutes at 4° C.Supernatants were passed through a 0.45 μm filter, and retained forassay. The bacterial cells were then washed twice in PBS, resuspended in1.5 ml of PBS and disrupted in a French pressure cell at 12,000 lb/in²to obtain a cell lysate (Fasano et al, Infect. Immun., 58:3717-3723(1991)). The cell lysate was then mixed with 3.5 ml of PBS (final volume5.0 ml), clarified by centrifugation at 18,000×g for 20 minutes at 4°C., and filter-sterilized using a 0.45 μm membrane.

Fractions of the culture supernatant of strain M4243 were obtained asdescribed in Section E. above.

Cytotoxicity assays were performed on the cell lysate and 3 differentculture supernatant fractions (less than 30 kDa, 30-100 kDa, and morethan 100 kDa), 10 with Vero cells by the method of Gentry et al, J.Clin. Microbiol., 12:361-366 (1980)). Serial two-fold dilutions (1:2 to1:64) of the culture supernatant fractions and cell lysate were tested,and the cytotoxic dose required to kill 50% of the Vero cells (CD50) wasestimated spectrophotometrically (Gentry et al, J. Clin. Microbiol.,12:361-366 (1980)).

Whole culture supernatants and cell lysates of enterohemorrhagic E. coli(EHEC) strain 933J, serotype 0157:H7, which elaborates Shiga-like toxin1 (SLT1), were used as the positive control in the Vero cellcytotoxicity assay (Fasano et al, Infect. Immun., 58:3717-3723 (1991)).The whole supernatant of non-pathogenic E. coli strains HS, which hasbeen used extensively as a negative control in assays of pathogenicityand in clinical studies (Levine et al, Lancet, I:1119-1122 (1978); andLevine et al, J. Infect. Dis., 148:699-709 (1983)), was used as anegative control in the Vero cell cytotoxicity assay.

Since the positive control (EHEC) killed more than 50% of the Vero cellsat a 1:64 dilution, a 10-fold dilution of both supernatants and lysatesfrom EHEC was tested. Cytotoxic titers were expressed as the reciprocalof the CD₅₀ /mg protein of the 30-100 kDa culture supernatant fractionor cell lysate; the protein content was measured by the method ofBradford, Anal. Biochem., 72:248-254 (1976)).

Both supernatant and lysate of the positive control strain EHEC strain933J serotype (0157:H7) showed a high level of cytotoxicity (0.5×10³ and3.4×10⁴ CD₅₀ /mg protein, respectively). In contrast, the supernatant ofHS, the negative control, showed no cytotoxic activity. Against thesetwo extremes, M4243 exhibited a low-level of cytotoxic activity whichwas restricted to the less than 30 kDa supernatant fraction (4.2×10²CD₅₀ /mg protein) and the cell lysate (5.1×10² CD₅₀ /mg protein).

The cytotoxic assay described above was repeated, except that HeLa cellswere substituted for Vero cells. As a result of this experiment, it wasdetermined that the 30-100 kDa fraction obtained from Shigella flexneri2a supernatant and cell lysate also does not possess any cytotoxicactivity against HeLa cells. On the other hand, as expected, andconsistent with the results obtained using Vero cells, only the lessthan 30 kDa supernatant fraction obtained from Shigella flexneri 2apossesses cytotoxic activity against HeLa cells (3.2×10² CD₅₀ /mgprotein). Also as expected, the cell lysate fraction from Shigellaflexneri 2a, which contains the less than 30 kDa fraction possessescytotoxic activity against HeLa cells (4.4×10² CD₅₀ /mg protein).

Thus, the enterotoxin (30-100 kDa fraction) activity and cytotoxin (lessthan 30 kDa fraction) activity found in Shigella flexneri 2a are theresult of two distinct moieties.

Hence, the enterotoxin appears to be responsible for the diarrheainduced by Shigella flexneri 2a, since the 30-100 kDa fraction (wherethe enterotoxic activity was localized) was responsible for fluidaccumulation in rabbit ileal loops and in electrical responses in Ussingchambers.

EXAMPLE 2 Preparation of Antisera

A. Preparation of Antibodies in Rabbits

1.0 ml of the 30-100 kDa fraction from the supernatant of Shigellaflexneri 2a strain M4243 that showed enterotoxic activity was mixed withan equal volume of Freund's complete adjuvant and inoculatedintramuscularly in four separate sites in male New Zealand whiterabbits. A booster dose (1.0 ml) was administered four weeks later, andone month thereafter the animals were bled to obtain antisera. Antiserato EIEC enterotoxin (EIET) from strain CVD/EI-34 (0136:H-) was preparedin the identical manner. These antisera are herein referred to asanti-Shigella flexneri 2a enterotoxins (anti-ShETs) andanti-enteroinvasive E. coli enterotoxin (anti-EIET).

B. Preparation of Antibodies in Humans

Pre- and post-challenged (convalescent) serum pools from 10 adultvolunteers who developed diarrhea after ingesting Shigella flexneri 2aM4243 (Kotloff et al, Infect. Immun., 60:2218-2224 (1992)) were preparedfor use in neutralization experiments in Ussing chambers (FIGS. 2C and2D), and for Western immunoblots (FIG. 4).

EXAMPLE 3 Purification and Partial Sequencing of Shigella Enterotoxin 1(ShET1)

A. Purification

Large-scale preparation of Shigella flexneri 2a enterotoxin wasundertaken in order to obtain sufficient material for furthercharacterization and analyses. Plasmid-cured S. flexneri 2a M4243avirwas used in order to avoid expression of both ShET2 and plasmid-encodedmembrane associated proteins (Hale et al, Infect. Immun., 50:620-6291985)) which are known to be similar in size to the fractions exhibitingenterotoxic activity and to be antigenic in volunteers (Van De Verg etal, J. Infect. Dis., 166:158-161 (1992)).

More specifically, plasmid-cured Shigella flexneri 2a was inoculatedinto 30 liters of L-broth containing 25 μg/ml of the iron-chelator,ethylenediamine-di-o-hydroxyphenylacetic acid (EDDA) (Rogers, Infect.Immun., Z:445-456 (1973)), and incubated overnight at 37° C. in the NewBrunswick Scientific 30 liter fermentor. Bacterial cells were removed bycentrifugation at 5,000×g in a Sharples industrial centrifuge, and thesupernatant was filtered through a 0.45 μm filter. This filtrate(approximately 30 liters) was fractionated to isolate and concentrate100-fold the moieties falling within the 30-100 kDa range as describedabove, except Pellicon tangential flow cassettes (Millipore) were usedfor ultrafiltration processing of these larger volumes. This filtrateexhibited enterotoxic activity similar to levels observed for smallerbatches employing the plasmid-cured strain.

A 10 ml aliquot of the 30-100 kDa concentrate was then furtherfractionated by replicate separations with an HPLC size exclusion column(SEC-2000, 7.5×600 cm with guard column, Phenomenex, Torrance, Calif.).Fractions were eluted from the column with PBS at 0.5 ml/min. Thefractions containing moieties in the 65-75 kDa range were collected,pooled and concentrated by vacuum dialysis to 1.0 ml employing a 10 kDamembrane (MicroProDiCon, Spectrum Medical Industries, Los Angeles,Calif.). An aliquot of this material was reserved for enterotoxin assay,and the remainder was separated by sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE) (Laemmli, Nature, 227:680-685 (1970))using an 11 cm preparative well with peripheral marker lanes. Theresultant 18 bands were transferred to a nitrocellulose membrane by themethod of Towbin et al, (Towbin et al, Proc. Natl. Acad. Sci. U.S.A.,76:4350-4354 (1979)).

Multiple 2 mm wide vertical strips of the nitrocellulose membrane wereprepared and stained with colloidal gold (Aurodye, JanssenPharmaceutica, Piscataway, N.J.) to visualize protein bands, or reactedwith the pooled convalescent sera by Western immunoblotting techniques(Vial et al, J. Infect. Dis., 158:70-79 (1988)).

Five protein bands were identified by the convalescent serum Westernstrips indicating their antigenic relatedness. The five protein bandswere aligned with the remainder of the nitrocellulose blot which hadbeen reversibly stained with Ponceau S (colloidal gold (Harlow et al,Antibodies: A Laboratory Manual, p. 494 (1988)). Using a scalpel, bandsof about 10 cm in length corresponding to immunoreactive material fromeach of the five protein bands were carefully excised by identificationand alignment with the Western and protein stained strips. Material fromeach of these bands were eluted (Montelero, Electrophoresis, 8:432-438(1987)) by dissolution of the nitrocellulose in 200 μl of dimethylsulfoxide, addition of four volumes of water to precipitate thenitrocellulose, followed by centrifugation at 10,000×g, and dialysis ofthe supernatant against PBS.

Each sample, in addition to the reserved 65-75 kDa sizing columnfraction, and material from a mock-blotted and extracted nitrocellulosestrip as positive and negative controls, respectively, was then testedfor enterotoxic activity in Ussing chambers, as discussed in Example 1above. The results are shown in FIG. 4.

As shown in FIG. 4, three of the bands, of approximate MW 63 kDa, 53 kDaand 41 kDa, exhibited enterotoxic activity. Replicates of a bandcorresponding to a MW of 41 kDa showed a consistent mean rise in I_(sc)of 70.4 μAmp/cm², whereas the 63 kDa and 53 kDa bands exhibited rises inI_(sc) of 24.3 and 19.5 μAmp/cm², respectively. The remaining twoimmunoreactive bands showed no enterotoxic activity.

The observation that convalescent sera from volunteers who were fedwild-type S. flexneri 2a contain antibodies that neutralize theenterotoxic activity S. flexneri 2a supernatants in Ussing chambers, andthat specifically bind to immobilized protein shown to produce suchactivity, demonstrates that ShET1 is expressed in vivo where it elicitsan immune response. Thus, it is likely that this enterotoxin plays arole in the pathogenesis of Shigella diarrhea in humans.

B. N-terminal Sequencing of ShET1

To obtain greater protein mass for sequencing, scale-up of thechromatographic procedure was preformed using Sephacryl S-200(Pharmacia, Piscataway, N.J.) packed in a calibrated, 4° C. jacketed,5×100 cm XK 50/100 column (Pharmacia). The 65-75 kDa size fraction washandled as above except that a polyvinylidine diflouridine membrane,Immobilon, Millipore) was substituted for nitrocellulose forelectrophoretic transfer. The three protein bands, identified asdescribed above, were excised, extensively rinsed with distilled waterand dried. Individual strips bearing the protein bands were thensubjected to N-terminal sequencing on an Applied Biosystems model 477Asequencer, as described by Hall et al, J. Bacteriol., 171:6372-6374(1989). The determined N-terminal sequence data are shown in Table 2below.

                                      TABLE 2                                     __________________________________________________________________________    Preliminary N-terminal amino acid sequence of Shigella enterotoxin 1                Proposed                                                                MW of A:B                                                                     enterotoxic                                                                         subunit                                                                            N-terminal amino acid sequence                                     moiety                                                                              ratio.sup.•                                                                  1.sup.‡                                                                2  3  4  5  6  7  8  9  10 11 12 13 14                         __________________________________________________________________________    63 kDa                                                                              A1:B3                                                                              Ala Pro                                                                              Pro                                                                              Val                                                                              (SEQ ID NO:3)                                                    Asp.sup.§                                                                    Thr   Leu                                                      53 kDa                                                                              A1:B2                                                                              Ala Pro                                                                              Pro                                                                              Val                                                                              (SEQ ID NO:3)                                                    Asp Thr   Leu                                                      41 kDa                                                                              A1:B1                                                                              Ala Pro                                                                              Pro                                                                              Val                                                                              Pro                                                                              Ile                                                                              Asn                                                                              Pro                                                                              Ala                                                                              Xaa                                                                              Pro                                                                              Ile                                                                              Xaa                                                                              Arg*                                  Asp Thr      Glu         Phe   Arg                                                                              Arg                              __________________________________________________________________________     .sup.• assuming an A subunit size of about 30 kDa and a B subunit       size of about 11 kDa                                                          .sup.‡ sequencing cycle number                                     .sup.§ Duplicate amino acid signals detected for samples at position     indicated                                                                     *(SEQ ID NO:4)                                                           

As shown in Table 2 above, a definitive extended sequence could not bedetermined from the material available for any of the three bands.However, the identical putative amino acid sequence was found for thefirst four residues of all three bands. Moreover, the data derivedsuggested that two distinct N-termini were being identified. Notably,this was consistent for all three bands examined.

The University of Wisconsin package (Genetics Computer Group, Madison,Wis.) (Devereux et al, Nucleic Acids Res., 12:387-395 (1984)), databases containing known protein sequences and untranslated DNA sequenceswere perused to identify those with potential amino acid homology to theputative N-terminal sequences acquired from the above samples. GenBankrelease 75.0 and PIR Protein 35.0 were also examined using the TFASTAand WORDSEARCH programs. No apparent regions of extensive alignment werefound to exist. In addition, no substantial homology to known bacterialtoxins was detected.

The common A:B_(n) active:binding unit motif frequently encountered inbacterial enterotoxins, including cholera toxin (CT) (LoSpalluto et al,Biochem. Biophys. Acta, 257:158-166 (1972)), heat-labile enterotoxin(LT) of enterotoxigenic E. coli (Clements et al, Infect. Immun.,38:806-809 (1982)) and Shiga toxin of S. dysenteriae 1 (Olsnes et al, J.Biol. Chem., 256:8732-8738 (1981); and Seidah et al, J. Biol. Chem.,261:13928-13931 (1986)), may be reflected in the above data. That is, asproposed in Table 2, the apparent molecular sizes of active material areconsistent with such stoichiometries based upon the sizes of the A(28-32 kDa) and B (7.7-11 kDa) subunits of the previously identifiedenterotoxins. By extension, a holotoxin consistent with a size of 65-75kDa and an A1:B4 structure would be predicted by these conventions.These tentative configurations also satisfy the usual requirements forboth a binding and an active domain that allow the enterotoxin to attachand gain entrance to enterocytes and to initiate events that culminatein intestinal secretion.

EXAMPLE 4 Gene Sequencing of Enteroinvasive E. coli Enterotoxin

A genetic approach was employed to identify and clone the enterotoxinfrom enteroinvasive E. coli. More specifically, TnphoA insertion mutantswere generated in EIEC strain EI-37 (0136:NM) (Fasano et al, Infect.Immun., 58:3717-3723 (1991)) as described by Taylor et al, J.Bacteriol., 171:1870-1978 (1989). The resulting TnphoA insertion mutantswere screened for increased expression of alkaline phosphatase in lowiron L-agar (containing 30 μg/ml of EDDA) compared with standard L-agar.As a result, nine insertion mutants with increased expression ofalkaline phosphatase were identified.

The supernatants from the resulting nine TnphoA insertion mutants werethen tested in Ussing chambers as described above, and two of themutants were found to have significantly less enterotoxic activity, asdefined by changes in I_(sc), than the wild-type parent, suggesting thatthe phoA gene was inserted into the open reading frame that encodesenterotoxic activity.

DNA was then purified from the two mutants, and the purified DNA wasdigested with BamHI. The resulting DNA fragments, which flank the TnphoAinsertions, were cloned into the BamHI site of vector pBluescript Sk+/-(Stratagene, La Jolla, Calif.). Then, the cloned DNA was hybridizedagainst a pHC79 cosmid library of EIEC strain EI-34 (Fasano et al,Infect. Immun., 58.:3717-3723 (1991)). The flanking DNA sequences fromone of the two TnphoA insertion mutants were found to be homologous tonine cosmid clones. Random subcloning of these cosmid clones intopBluescript Sk+/- led to the identification of a 2.8 kb HindIII fragmentwhich was found to encode enterotoxin activity in Ussing chambers. Thisfragment, when cloned into the HindIII site of pBluescript Sk+/-, gaverise to pJS26 (FIG. 5). DH5α (Gibco/BRL Life Technologies, Gaithersberg,Md.) was transformed with pJS26, and found to confer reproducibleincreases in I_(sc) in Ussing chambers.

The 2.8 kb HindIII fragment was manually sequenced, and two potentialopen reading frames (orf's), encoding predicted peptides of 62.8 kDa and16.1 kDa were found (FIG. 5).

The 2.8 kb HindIII fragment was digested with ClaI and subcloned intoHindIII- and ClaI-digested pBluescript Sk+/-, to give rise to pJS264,which contained only the 62.8 kDa orf (FIG. 5). DH5α transformed withpJS264 exhibited rises in I_(sc) in Ussing chambers similar to thatfound with the entire 2.8 kb HindIII fragment. This orf, whose DNAsequence, along with the determined amino acid sequence are shown inFIGS. 6A-6C (SEQ ID NO:1), was therefore designated tie (for "toxininvasive E. coli").

The 2.8 kb HindIII fragment was also digested with ClaI and subclonedinto HindIII- and ClaI-digested pBluescript Sk+/-, to give rise topJS263, which contained only the 16.1 kDa orf (FIG. 5). DH5α transformedwith pJS264 did not elicit rises in I_(sc) in Ussing chambers.

A GenBank search for amino acid homology of the translated orf'srevealed no significant identity to any known prokaryotic sequences.

The 2.8 kb HindIII fragment containing the tie gene was then digestedwith AccI and cloned into DH5α so as to obtain pJS261 (FIG. 5), whichwas then used to transform DH5α. The resulting transformant was alsofound to express enterotoxic activity when tested in Ussing chambers asdescribed above.

In order to gauge the effect of the tie gene on secretory activity, adeletion mutation was constructed by digesting the tie gene in pJS26with NdeI and SphI. The resulting plasmid was designated pJS26a (FIG.5). This plasmid lacked the first two-thirds of the N-terminus of theopen reading frame. This plasmid was then used to transform DH5α, andtested in Ussing chambers as described above. The supernatant obtainedfrom the pJS26Δ transformants elicited less response in the Ussingchamber assay when compared to pJS26, confirming that tie gene is theEIET structural gene.

Thus, unlike ShET1, which as discussed above is believed to be composedof A and B subunits, EIET is a single molecule.

EXAMPLE 5 Gene Sequencing of Shigella Enterotoxin 2 (ShET2)

As discussed above, Shigella and EIEC share some similarities. Thus, theorf containing the gene encoding the EIEC enterotoxin shown in FIGS.6A-6D (SEQ ID NO:1) was used as a probe to determine whether Shigellahas similar DNA sequences.

More specifically, purified genomic DNA was obtained from each of S.flexneri 5a M4243 and S. flexneri 2a M4243avir, digested with SalI,another screened for hybridization with the tie gene. The DNA-DNAhybridization showed the presence of a single 3.5 kb band in genomic DNAfrom the wild-type strain, but not from the plasmid-cured derivative.This result suggests that the homologous DNA is located on theinvasiveness plasmid.

The 3.5 kb SalI fragment was identified on the S. flexneri 2a M4243plasmid by PCR using the following oligonucleotide primers thathybridize to the tie gene (CAGTGTATCACCACGAG (SEQ ID NO:13); andAAATTATCTACAGTCAG (SEQ ID NO:14)), and sequenced using an automatedsequencer. The resulting DNA sequence, along with the determined aminoacid sequence are shown in FIGS. 7A-7D (SEQ ID NO:2). As shown in FIGS.7A-7D (SEQ ID NO:2), this fragment was found to contain a 1595 bp openreading frame and has at least 99% homology to the EIET gene. ThisShigella gene encodes for a protein of a predicted MW of 63 kDa, and apI of 6.36. No leader peptide was identified. The analysis of thepeptide structure revealed three possible membrane spanning domains(amino acid positions 120-140, 260-300 and 480-520) and five cysteineresidues. A predicted ribosome binding site is found at nucleotidepositions 290-293. When the translation of this open reading frame wascompared to the N-terminal sequence of ShET1 shown in Table 2, nohomologies were found, suggesting that this gene, located on the S.flexneri 2a M4243 plasmid, encodes for a toxin (hereinafter named"ShET2") which is distinct from ShET1, but substantially identical toEIET.

Due to the similarity between the EIET gene and the ShET2 gene, it isevident that the gene located on S. flexneri 2a M4243 plasmid, i.e.,that hybridized with EIET gene probe, is the ShET2 structural gene.

EXAMPLE 6 Use of EIEC Enterotoxin Gene as a DNA Probe

The tie gene was used as a DNA probe and hybridized against a collectionof EIEC and Shigella strains under high stringency by the colony blotmethod. The results are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                        Prevalence of tie Gene in E. coli and Shigella                                Colony Blot Hybridization with tie Probe                                      Category   Positive    Negative % Positive                                    ______________________________________                                        Shigella   27          7        80%                                           EIEC       60          20       75%                                           Other E. coli                                                                            0           110       0%                                           ______________________________________                                    

As shown in Table 3 above, the tie-homologous sequences are present in80% (27/34) of Shigella strains, including members of all four Shigellaspecies (flexneri, boydii, sonnei and dysenteriae), and 75% of EIEC.None of 110 E. coli other than EIEC carried homologous sequences.

EXAMPLE 7 Gene Sequencing of Shigella Enterotoxin 1 (ShET1)

A colony immunoblot technique was utilized to clone the ShET1 gene(set1) using the rabbit polyclonal antibodies described in Example 2.

More specifically, a library of genomic DNA obtained from theplasmid-cured derivative of S. flexneri 2a strain 2457T, designated asstrain 2457TA (the Walter Reed Army Institute of Research), was obtainedby partial digestion with Sau3A. The resulting 5 to 10 kb fragments werepurified by GeneClean, and then Sau3A DNA termini were partially filledin with dATP and dGTP in a Klenow reaction.

Separately, the cos ends of undigested λZAPII vector (Stratagene, LaJolla, Calif.) were ligated, the vector digested with XhoI and theresulting termini partially filled in with dCTP and dTTP. This resultedin compatible ends between the vector and genomic inserts, but notbetween themselves.

The compatible ends of the genome fragments and the vector were ligatedand packaged using the Gigapack II Gold packing extract (Stratagene)system following the procedures recommended by the manufacturer. Theresulting λZAPII::2457TA library was titrated in E. coli strain XL1-BlueMRF' (Stratagene) to obtain a concentration of 100 plaques/100 mm plate.Next, the plaques were blotted with IPTG-saturated nitrocellulosefilters using the procedures for immunological screening of expressionof bacteriophage λ vector libraries described by Sambrook et al,Molecular Cloning. A Laboratory Manual, Second Edition, Cold SpringHarbor Laboratory Press (1989).

Then, 40 filters (approx. 4×10³ plaques) were screened with the rabbitpolyclonal antiserum described in Example 2, and six plaques were foundto be strongly positive. These plaques were harvested, and pBluescriptSk+/- containing the corresponding 2457TA DNA inserts were excised fromthe λZAPII vector using the ExAssist/SOLR system (Stratagene) usingprocedures recommended by the manufacturer.

The resulting pBluescript Sk+/- was used to infect DH5α, and 24 singlecolonies derived from each immunoblot-positive plaque were grown in 300ml of Fe⁺⁺ -depleted LB medium with 100 μg/ml ampicillin in 96-wellmicrotiter plates and cultured at 37° C. for 48 h. The supernatants ofthese cultures were then passed by gravity through nitrocellulose paperin a 96-well manifold (Biorad), and immunoblotted with the abovedescribed rabbit antiserum. The supernatants from clones derived fromone positive plaque were found to be strongly reactive.

Filter-sterilized supernatants from 6 arbitrarily-selected of thesestrongly reactive clones were tested on rabbit ileal mucosa in Ussingchambers. One of these supernatants induced I_(sc) changes (58.7+/-7.9μAmp/cm²) significantly higher then DH5α (17.9+/-7.3 μAmp/cm²) negativecontrol supernatants and equivalent to 2457TA supernatant (38.8+/-10.1μAmp/cm²). The plasmid contained in this clone, designated pF9-1-90, waspurified, mapped and a 6.0 kb DNA insert was found (see FIG. 8). Westernimmunoblots of supernatants from clones containing plasmid pF9-1-90showed the expression of similar banding pattern present in 2457TA, butnot in the host DH5α (pBluscript Sk+/-) alone.

Using the multiple restriction enzymes found in the polylinker ofpBluscript Sk+/- as reference, various segments of the 6.0 kb insertwere subcloned in the same vector. Supernatants from clones containingsegments of various sizes were tested in Ussing chambers andimmunoblots.

Single strand sequencing of a selected genomic insert in pF9-1-90 wasperformed by automated fluorescent sequencing (Applied Biosystems DNAsequencer Model 373A, Foster City, Calif.). The complementary DNA strandwas sequenced by chain-termination sequencing using the SequenaseVersion 2.0 DNA sequencing kit (USB, Cleveland, Ohio). Chain-terminationsequencing was used as well to identify and determine the orientation ofthe set1 genes in pset1, described below.

Sequencing analysis of a 3.0 kb DNA segment downstream of the promoterT7 in pF9-1-90 revealed two open reading frames (orf), of respectively146 bp (set1B) and 574 bp (set1A), in the same orientation, separated byonly 6.0 bp (FIGS. 9A-9B; SEQ ID NO:15).

Surprisingly, the ShET1 predicted amino acid sequence based on the DNAsequence shown in FIGS. 9A-9B did not corrspond to the N-terminal aminoacid sequence shown in Table 2. This confirms the difficulty in cloningthe ShET1 gene.

The predicted molecular weights (MW) of the protein molecules encoded bythese orfs are of approximately 7.0 kDa and 20 kDa for set1B and set1A,respectively. The finding of a 55 kDa protein in the immunoblotexperiments described below supports the concept of an A₁ :B₅configuration for the holotoxin, where the A subunit is 20 kDa and eachindividual B subunit is 7.0 kDa. The set1B gene has an upstream promotergoverning the transcription of both the set1B and set1A genes.

Analysis of the amino acid sequence of set1B revealed a peptidestructure with a predicted signal sequence. Comparison of the predictedprotein with the EMBL/GenBank library of sequences did not showsignificant homologies among prokariotic or eukariotic sequences at theamino acid or nucleotide level. The set1A gene has its own ShineDelgarno sequence 15 bp upstream the initiation codon. The predictedamino acid sequence of set1A also features a putative signal sequence.Comparison of this orf with the EMBL/GenBank did not reveal significanthomologies with known sequences.

A 1,093 bp fragment containing the set1 orfs (with an upstream segmentof 98 bp) was obtained by digesting the 6.0 Kb insert in pF9-1-90 withXmaI and cloning it in pBluescript SK+/-. The plasmid so obtained, namedpset1, was transformed into DH5α. DH5α(pset1) supernatant was thenimmunoblotted as described above, and tested in Ussing chambers forenterotoxic activity.

Immunoblot of the Fe⁺⁺ -depleted supernatant from the DH5α(pset1)culture revealed the expression of the 55 kDa protein band detected inS. flexneri 2a strain 2457TA and pF9-1-90 supernatants, but not in theDH5α negative control. DH5α(pset1) supernatant induced an increase inI_(sc) when tested in Ussing chambers (79.18+/-14.1 μAmp/cm² ; n=6)higher than that seen with S. flexneri 2a wild-type strain 2457TA(38.80+/-7.6 μAmp/cm² ; n=6) and DH5α(pF9-1-90) (53.63+/-11.3 μAmp/cm² ;n=8). All ShET1-containing supernatants tested in Ussing chambers showeda high increase of I_(sc) as compared to the changes induced bysupernatants obtained from the DH5α(pBluescript SK+/-) negative control(10.18+/8.5 μAmp/cm² ; n=7; p<0.01). The enterotoxic effect wasproportional to the level of expression of ShET1(pset1>pF9-1-90>2457TA), suggesting a dose-response relationship for thetoxicity of ShET1.

EXAMPLE 8 Construction of the Attenuated S. flexneri Strain CVD1203

S. flexneri 2a strain 2457T (Kotloff et al, Infect. Immun. 60:2218-2224(1992)), known to be virulent based on experimental challenge studies inadult volunteers, was selected as the wild-type parent to be attenuatedby introduction of a deletion in both the aroA and VirG genes.

More specifically, the aroA gene (Duncan et al, FEBS, 170:59-63 (1984))was subjected to polymerase chain reactions in a Programmable ThermalController unit, using Taq polymerase and buffer obtained from Promegato obtain a deletion of 201 nucleotides in the aroA gene, whichcorresponds to a deletion of amino acids 168-231 of the encoded enzyme.In particular, the 5' end of the aroA gene was amplified with theupstream primer (TAATCGAATTCATGGAATCCCTGACGTTA) (SEQ ID NO:5) so as tointroduce an EcoRI site, and with the down stream primer(GGTACCCCCAATATTAGGGCCATCAACGTCAACGTTGCCGCC) (SEQ ID NO:6) so as tointroduce KpnI and SspI sites. The 3' end of the aroA gene was amplifiedwith the upstream primer (AATATTGGGGGTACCGGTACTTATTTGGTCGAAGGCGATGCA)(SEQ ID NO:7) so as to introduce SspI and KpnI sites, and with thedownstream primer (TGATAAGTCGACTCAGGCTGCCTGGCTAAT) (SEQ ID NO:8) so asto introduce a SalI site. Both segments were amplified for 30 cycles of1 min at 94° C. 2 min at 50° C. and 4 min at 72° C.

In a second PCR reaction, the 5' and 3' segments were fused, and theresulting fusion product was amplified in the same reaction. In thisreaction, the given homologous regions (SspI-KpnI) annealed, effectivelyfusing the 5' and 3' segments, which at that time may have acted astheir own primers and/or templates for the Taq polymerase, dependingupon which stands of DNA were annealed. To facilitate this fusion, thefirst 15 cycles had an annealing temperature slope (1° C./8 sec from 40°C. to 50° C.+50° C. for 2 min), followed by 15 cycles with an annealingtemperature of 55° C. in which the new ΔaroA gene was amplified. TheΔaroA gene of Shigella was cloned into the EcoRI and SalI sites of thetemperature-sensitive vector pIB307 (Blomfield et al, Mol., Microbiol.,5:1447-1457 (1991)) to give rise to pIB307::ΔaroA. pIB307::ΔaroA waselectroporated into E. coli DH5α and grown at 30° C. In a second step,the sacB-neomycin^(R) segment of pIB279 (Blomfield et al, Mol.,Microbiol., 5:1447-1457 (1991)) was transferred into the BamHIpolylinker site of pIB307::ΔaroA, and the resultant plasmid, designatedpFJ201, was introduced into DH5α by electroporation, and incubated at30° C.

pFJ201 was electroporated into S. flexneri 245T to achieve allelicexchange in the wild-type strain. Co-integates representing a singlehomologous recombination were readily obtained. Using counter selection(Aro-sucrose plates at 30° C.), a clone was identified that hadcharacteristics of the double homologous recombination event, i.e.,representing allelic exchange of ΔaroA for aroA in the chromosome. Thisclones was kanamycin-sensitive, Congo red-positive, agglutinated with S.flexneria 2a antiserum, and was unable to grow in Shigella minimummedium (SMM) consisting of 0.4 g NaCl, 8.4 g K₂ HPO₄, 3.6 g KH₂ PO₄, 0.8g (NH₄)₂ SO₄, 2.5 g glucose, 0.05 g nicotonic acid, 0.05 g asparticacid, 0.05 g serine and 15 g nobel L-agar. SMM allows one to screen forΔaroA mutants colonies that cannot synthesize aromatic compounds denovo, and thus require exogenus aromatic compounds in order to grown.PCR of this strain demonstrated that the gene produced harbored adeletion; the wild-type product was 1.2 kb, whereas the product of theclone was 1.0 kb. Confirmation of the deletion was made using a 40 basesynthetic oligonucleotide sequence derived from the deleted portion ofthe gene. The 32P-labelled probe hybridized with wild-type colonies, butnot with the clone. This ΔaroA clone was designated CVD1201.1.

Strains ΔaroA CVD 1201.1 and wild-type 2457T were grown shaking at 37°C. in 5.0 ml volumes of SMM that was progressively supplemented witharomatic amino acids (50 mg L-tryptophan, 50 mg L-tyrosine, 50 mgL-phenylalanine), 10 mg ferric ammonium acetate and 10 mg PABA. CVD1201.1 required the addition of tryosine, tryptophan, phenylalanine andPABA in order to grow.

A deletion of 900 nucleotides in the virG gene (Lett et al, J.Bacteriol., 172:352-359 (1989)), which corresponds to a deletion ofamino acids 341-640 of the 120 kDa VirG protein, was obtained byfollowing steps analogous to that used for preparing the ΔaroA mutation.The specific engineered site for this deletion in the 120 kDa proteinrepresents a highly hydrophobic, poorly antigenic portion of themolecule according to the Jameson/Wolf antigenic index (IBI PustellSequence Analysis Programs). More specifically, the 5' end of the virGgene was amplified with the upstream primer(GGGGAATTCCAAATTCACAAATTTTTTTGT) (SEQ ID NO:9) so as to introduce anEcoRI site, and with the downstream primer(TCCATGCCATTCATGGAGTATTAATGAATT) (SEQ ID NO:10). The 3' end of the virGgene was amplified with the upstream primer(CTCCATGAATGGCATGGAAAGGCGGAATA) (SEQ ID NO:11), and the downstreamprimer (CGGGTCGACTCAGAAGGTATATTTCACACCCAA) (SEQ ID NO:12) so as tointroduce a SalI site. Amplification and fusion of the virG 5' and 3'segments were performed using the same PCR cycles described above. Theresulting new ΔvirG gene was cloned into the EcoRI and SalI sites of thepir-based suicide vector pKTN701 (Hone et al, Vaccine, 9:810-816(1991)), giving rise to pShΔvirG, which was electroporated into E. colistrain SY327 (Miller et al, J. Bacteriol., 170:2575-2583 (1983)). Theplasmid was then electroporated into strain Sm10λpir (Miller et al, J.Bacteriol., 170:2575-2583 (1983)). Sm10λpir(pShΔvirG) was used toconjugate the deletion cassette into the ΔaroA strain, CVD1201.1.

Suicide vector pShΔvirG was integrated into the virulence plasmid(ΔvirG) loci of the ΔaroA strain, CVD1201.1, to introduce the ΔvirGmutation by homologous recombination, followed bychloramphenicol-sensitive enrichment using the procedures described forSalmonella by Hone et al, Vaccine, 9:810-816 (1991).

An antibiotic-sensitive clone representing a putative successful doublehomologous recombination event was confirmed by PCR, Congo redpositivity, agglutination with S. flexneri 2a antiserum and failure tohybridize with the oligonucleotide probe specific for the deletedsequence.

In this manner the ΔaroA ΔVirG Shigella flexneri 2a mutant, CVD1203(ATCC No. 55556), was isolated.

The 120 kDa VirG protein was not detected in immunoblots using wholecell lysates of CVD1203, and a rabbit antiserum developed against theVirG peptide (Ile 359--Cys 375) representing a fraction of ΔVirG withinthe deleted portion of ΔVirG. However, an 85 kDa band was detected whenrabbit antiserum against another VirG peptide (Leu 55--Thr 73),representing a portion of ΔVirG that it expressed in CVD1203, was usedin the immunoblot.

CVD1203, like its wild-type parent, grow on enteric media, which containsufficient PABA and aromatic amino acids, and manifest a typical acidbutt/alkaline slant reaction with H₂ S or gas 18-24 h after inoculationof triple sugar iron agar slants. A silver-strained SDS-PAGE of LPS fromstrains 2457T and CVD1203 demonstrated the identity of the LPS pattern.Similarly, a Western immunoblot of LPS from CVD1203 and 2457T thatreacted with human antisera to Shigella flexneri 2a 2457T showedidentical bands irrespective of the source of the LPS preparation. Waterextracts of CVD1203 and 2457T exhibited identical single bands onWestern immunoblots with monoclonal antibodies to either IpaB (42 kDa)or to IpaC (62 kDa). Using anti-IpaC monoclonal antibody, dotimmunoblots of serial dilutions of the two extracts containing equalamounts of protein demonstrated the same endpoints, indicating that bothstrains produced the same amount of IpaC.

While the invention has been described in detail, and with reference tospecific embodiments thereof, it will be apparent to one of ordinaryskill in the art that various changes and modifications can be madetherein without departing from the spirit and scope thereof.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 15                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2008 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: genomic DNA                                               (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Enteroinvasive E. coli                                          (B) STRAIN: EI-37 (0136:NM)                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ATCGATATATTGTTTATTGTCAGTATGGCTCAATGTGATAATAGTTGGAAAGTTTGATGG60                GTTTCGCCCCGTTGTAGCGGTAGTCGACCCCGTTGTAGCGGTAGTCGAGCTGGAAGGTCT120               TCAGGCACTGCTTACAGCGATAGAGCAGCCCCCCAGAACTGGAATGGCCGTTCCGATACC180               CCCCTGAGTTTCAGAGTAACGGGGACAAACCACATCAATCTTTGCCATCAATCATCCAAA240               GGGCAAAGAGTACAACAACACTAAGTCTGCGTCACAACCCATCAATGAAAGGAATATATA300               CATATGCCATCAGTAAATTTAATCCCATCAAGGAAAATATGTTTGCAA348                           MetProSerValAsnLeuIleProSerArgLysIleCysLeuGln                                 151015                                                                        AATATGATAAATAAAGACAACGTCTCTGTTGAGACAATCCAGTCTCTA396                           AsnMetIleAsnLysAspAsnValSerValGluThrIleGlnSerLeu                              202530                                                                        TTGCACTCAAAACAATTGCCATATTTTTCTGACAAGAGGAGTTTTTTA444                           LeuHisSerLysGlnLeuProTyrPheSerAspLysArgSerPheLeu                              354045                                                                        TTAAATCTAAATTGCCAAGTTACCGATCACTCTGGAAGACTTATTGTC492                           LeuAsnLeuAsnCysGlnValThrAspHisSerGlyArgLeuIleVal                              505560                                                                        TGTCGACATTTAGCTTCCTACTGGATAGCACAGTTTAACAAAAGTAGT540                           CysArgHisLeuAlaSerTyrTrpIleAlaGlnPheAsnLysSerSer                              657075                                                                        GGTCACGTGGATTATCATCACTTTGCTTTTCCGGATGAAATTAAAAAT588                           GlyHisValAspTyrHisHisPheAlaPheProAspGluIleLysAsn                              80859095                                                                      TATGTTTCAGTGAGTGAAGAAGAAAAGGCTATTAATGTGCCTGCTATT636                           TyrValSerValSerGluGluGluLysAlaIleAsnValProAlaIle                              100105110                                                                     ATTTATTTTGTTGAAAACGGTTCATGGGGAGATATTATTTTTTATATT684                           IleTyrPheValGluAsnGlySerTrpGlyAspIleIlePheTyrIle                              115120125                                                                     TTCAATGAAATGATTTTTCATTCCGAAAAAAGCAGAGCACTAGAAATA732                           PheAsnGluMetIlePheHisSerGluLysSerArgAlaLeuGluIle                              130135140                                                                     AGTACATCAAATCACAATATGGCATTAGGCTTGAAGATTAAAGAAACT780                           SerThrSerAsnHisAsnMetAlaLeuGlyLeuLysIleLysGluThr                              145150155                                                                     AAAAATGGGGGGGATTTTGTCATTCAGCTTTATGATCCCAACCATACA828                           LysAsnGlyGlyAspPheValIleGlnLeuTyrAspProAsnHisThr                              160165170175                                                                  GCAACTCATTTACGAGCAGAGTTTAACAAATTTAACTTAGCTAAAATA876                           AlaThrHisLeuArgAlaGluPheAsnLysPheAsnLeuAlaLysIle                              180185190                                                                     AAAAAACTGACTGTAGATAATTTTCTTGATGAAAAACATCAGAAATGT924                           LysLysLeuThrValAspAsnPheLeuAspGluLysHisGlnLysCys                              195200205                                                                     TATGGTCTTATATCCGACGGTATGTCTATATTTGTGGACAGACATACT972                           TyrGlyLeuIleSerAspGlyMetSerIlePheValAspArgHisThr                              210215220                                                                     CCAACAAGCATGTCCTCCATAATCAGATGGCCTAATAATTTACTTCAC1020                          ProThrSerMetSerSerIleIleArgTrpProAsnAsnLeuLeuHis                              225230235                                                                     CCCAAAGTTATTTATCACGCGATGCGTATGGGATTGACTGAGCTAATC1068                          ProLysValIleTyrHisAlaMetArgMetGlyLeuThrGluLeuIle                              240245250255                                                                  CAAAAAGTAACAAGAGTCGTACAACTATCTGACCTTTCAGACAATACG1116                          GlnLysValThrArgValValGlnLeuSerAspLeuSerAspAsnThr                              260265270                                                                     TTAGAATTACTTTTGGCAGCCAAAAATGACGATGGTTTGTCAGGATTG1164                          LeuGluLeuLeuLeuAlaAlaLysAsnAspAspGlyLeuSerGlyLeu                              275280285                                                                     CTTTTAGCTTTACAAAATGGGCATTCAGATACAATCTTAGCATACGGA1212                          LeuLeuAlaLeuGlnAsnGlyHisSerAspThrIleLeuAlaTyrGly                              290295300                                                                     GAACTCCTGGAAACTTCTGGACTTAACCTTGATAAAACGGTAGAACTA1260                          GluLeuLeuGluThrSerGlyLeuAsnLeuAspLysThrValGluLeu                              305310315                                                                     CTAACTGCGGAAGGAATGGGAGGACGAATATCGGGTTTATCCCAAGCA1308                          LeuThrAlaGluGlyMetGlyGlyArgIleSerGlyLeuSerGlnAla                              320325330335                                                                  CTTCAAAATGGGCATGCAGAAACTATCAAAACATACGGAAGGCTTCTC1356                          LeuGlnAsnGlyHisAlaGluThrIleLysThrTyrGlyArgLeuLeu                              340345350                                                                     AAGAAGAGAGCAATAAATATCGAATACAATAAGCTGAAAAATTTGCTG1404                          LysLysArgAlaIleAsnIleGluTyrAsnLysLeuLysAsnLeuLeu                              355360365                                                                     ACCGCTTATTATTATGATGAAGTACACAGACAGATACCTGGACTAATG1452                          ThrAlaTyrTyrTyrAspGluValHisArgGlnIleProGlyLeuMet                              370375380                                                                     TTTGCTCTTCAAAATGGACATGCAGATGCTATACGCGCATACGGTGAG1500                          PheAlaLeuGlnAsnGlyHisAlaAspAlaIleArgAlaTyrGlyGlu                              385390395                                                                     CTCATTCTTAGCCCCCCTCTCCTCAACTCAGAGGATATTGTAAATTTG1548                          LeuIleLeuSerProProLeuLeuAsnSerGluAspIleValAsnLeu                              400405410415                                                                  CTGGCCTCAAGGAGATATGACAATGTTCCCGGACTTCTGTTAGCATTG1596                          LeuAlaSerArgArgTyrAspAsnValProGlyLeuLeuLeuAlaLeu                              420425430                                                                     AATAATGGACAGGCTGATGCAATCTTAGCTTATGGTGATATCTTGAAT1644                          AsnAsnGlyGlnAlaAspAlaIleLeuAlaTyrGlyAspIleLeuAsn                              435440445                                                                     GAGGCAAAACTTAACTTGGATAAAAAAGCAGAGCTGTTAGAAGCGAAA1692                          GluAlaLysLeuAsnLeuAspLysLysAlaGluLeuLeuGluAlaLys                              450455460                                                                     GATTCTAATGGTTTATCTGGATTGTTTGTAGCCTTGCATAATGGATGT1740                          AspSerAsnGlyLeuSerGlyLeuPheValAlaLeuHisAsnGlyCys                              465470475                                                                     GTAGAAACAATTATTGCTTATGGGAAAATACTTCACACTGCAGACCTT1788                          ValGluThrIleIleAlaTyrGlyLysIleLeuHisThrAlaAspLeu                              480485490495                                                                  ACTCCACATCAGGCATCAAAATTACTGGCAGCAGAAGGCCCAAATGGG1836                          ThrProHisGlnAlaSerLysLeuLeuAlaAlaGluGlyProAsnGly                              500505510                                                                     GTATCTGGATTAATTATAGCTTTTCAAAATAGGAATTTTGAGGCAATA1884                          ValSerGlyLeuIleIleAlaPheGlnAsnArgAsnPheGluAlaIle                              515520525                                                                     AAAACTTATATGGGAATAATAAAAAATGAAAATATTACACCTGAAGAA1932                          LysThrTyrMetGlyIleIleLysAsnGluAsnIleThrProGluGlu                              530535540                                                                     ATAGCAGAACACTTGGACAAAAAAAATGGAAGTGATTTTCTAGAAATT1980                          IleAlaGluHisLeuAspLysLysAsnGlySerAspPheLeuGluIle                              545550555                                                                     ATGAAGAATATAAAAAGCTGAATATTAT2008                                              MetLysAsnIleLysSer                                                            560565                                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1722 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: genomic DNA                                               (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Shigella flexneri 2a                                            (B) STRAIN: M4243                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       ACCCATCAATGAAAGGAATATATACATATGCCATCAGTAAATTTAATCCCA51                         MetProSerValAsnLeuIlePro                                                      15                                                                            TCAAGGAAAATATGTTTGCAAAATATGATAAATAAAGACAACGTCTCT99                            SerArgLysIleCysLeuGlnAsnMetIleAsnLysAspAsnValSer                              101520                                                                        GTTGAGACAATCCAGTCTCTATTGCACTCAAAACAATTGCCATATTTT147                           ValGluThrIleGlnSerLeuLeuHisSerLysGlnLeuProTyrPhe                              25303540                                                                      TCTGACAAGAGGAGTTTTTTATTAAATCTAAATTGCCAAGTTACCGAT195                           SerAspLysArgSerPheLeuLeuAsnLeuAsnCysGlnValThrAsp                              455055                                                                        CACTCTGGAAGACTTATTGTCTGTCGACATTTAGCTTCCTACTGGATA243                           HisSerGlyArgLeuIleValCysArgHisLeuAlaSerTyrTrpIle                              606570                                                                        GCACAGTTTAACAAAAGTAGTGGTCACGTGGATTATCATCACTTTGCT291                           AlaGlnPheAsnLysSerSerGlyHisValAspTyrHisHisPheAla                              758085                                                                        TTTCCGGATGAAATTAAAAATTATGTTTCAGTGAGTGAAGAAGAAAAG339                           PheProAspGluIleLysAsnTyrValSerValSerGluGluGluLys                              9095100                                                                       GCTATTAATGTGCCTGCTATTATTTATTTTGTTGAAAACGGTTCATGG387                           AlaIleAsnValProAlaIleIleTyrPheValGluAsnGlySerTrp                              105110115120                                                                  GGAGATATTATTTTTTATATTTTCAATGAAATGATTTTTCATTCCGAA435                           GlyAspIleIlePheTyrIlePheAsnGluMetIlePheHisSerGlu                              125130135                                                                     AAAAGCAGAGCACTAGAAATAAGTACATCAAATCACAATATGGCATTA483                           LysSerArgAlaLeuGluIleSerThrSerAsnHisAsnMetAlaLeu                              140145150                                                                     GGCTTGAAGATTAAAGAAACTAAAAATGGGGGGGATTTTGTCATTCAG531                           GlyLeuLysIleLysGluThrLysAsnGlyGlyAspPheValIleGln                              155160165                                                                     CTTTATGATCCCAACCATACAGCAACTCATTTACGAGCAGAGTTTAAC579                           LeuTyrAspProAsnHisThrAlaThrHisLeuArgAlaGluPheAsn                              170175180                                                                     AAATTTAACTTAGCTAAAATAAAAAAACTGACTGTAGATAATTTTCTT627                           LysPheAsnLeuAlaLysIleLysLysLeuThrValAspAsnPheLeu                              185190195200                                                                  GATGAAAAACATCAGAAATGTTATGGTCTTATATCCGACGGTATGTCT675                           AspGluLysHisGlnLysCysTyrGlyLeuIleSerAspGlyMetSer                              205210215                                                                     ATATTTGTGGACAGACATACTCCAACAAGCATGTCCTCCATAATCAGA723                           IlePheValAspArgHisThrProThrSerMetSerSerIleIleArg                              220225230                                                                     TGGCCTGATAATTTACTTCACCCCAAAGTTATTTATCACGCGATGCGT771                           TrpProAspAsnLeuLeuHisProLysValIleTyrHisAlaMetArg                              235240245                                                                     ATGGGATTGACTGAGCTAATCCAAAAAGTAACAAGAGTCGTACAACTA819                           MetGlyLeuThrGluLeuIleGlnLysValThrArgValValGlnLeu                              250255260                                                                     TCTGACCTTTCAGACAATACGTTAGAATTACTTTTGGCAGCCAAAAAT867                           SerAspLeuSerAspAsnThrLeuGluLeuLeuLeuAlaAlaLysAsn                              265270275280                                                                  GACGATGGTTTGTCAGGATTGCTTTTAGCTTTACAAAATGGGCATTCA915                           AspAspGlyLeuSerGlyLeuLeuLeuAlaLeuGlnAsnGlyHisSer                              285290295                                                                     GATACAATCTTAGCATACGGAGAACTCTTGGAAACTTCTGGACTTAAC963                           AspThrIleLeuAlaTyrGlyGluLeuLeuGluThrSerGlyLeuAsn                              300305310                                                                     CTTGATAAAACGGTAGAACTACTAACTGCGGAAGGAATGGGAGGACGA1011                          LeuAspLysThrValGluLeuLeuThrAlaGluGlyMetGlyGlyArg                              315320325                                                                     ATATCGGGTTTATCCCAAGCACTTCAAAATGGGCATGCAGAAACTATC1059                          IleSerGlyLeuSerGlnAlaLeuGlnAsnGlyHisAlaGluThrIle                              330335340                                                                     AAAACATACGGAAGGCTTCTCAAGAAGAGAGCAATAAATATCGAATAC1107                          LysThrTyrGlyArgLeuLeuLysLysArgAlaIleAsnIleGluTyr                              345350355360                                                                  AATAAGCTGAAAAATTTGCTGACCGCTTATTATTATGATGAAGTACAC1155                          AsnLysLeuLysAsnLeuLeuThrAlaTyrTyrTyrAspGluValHis                              365370375                                                                     AGACAGATACCCGGACTAATGTTTGCTCTTCAAAATGGACATGCAGAT1203                          ArgGlnIleProGlyLeuMetPheAlaLeuGlnAsnGlyHisAlaAsp                              380385390                                                                     GCTATACGCGCATACGGTGAGCTCATTCTTAGCCCCCCTCTCCTCAAC1251                          AlaIleArgAlaTyrGlyGluLeuIleLeuSerProProLeuLeuAsn                              395400405                                                                     TCAGAGGATATTGTAAATTTGCTGGCCTCAAGGAGATATGACAATGTT1299                          SerGluAspIleValAsnLeuLeuAlaSerArgArgTyrAspAsnVal                              410415420                                                                     CCCGGACTTCTGTTAGCATTGAATAATGGACAGGCTGATGCAATCTTA1347                          ProGlyLeuLeuLeuAlaLeuAsnAsnGlyGlnAlaAspAlaIleLeu                              425430435440                                                                  GCTTATGGTGATATCTTGAATGAGGCAAAACTTAACTTGGATAAAAAA1395                          AlaTyrGlyAspIleLeuAsnGluAlaLysLeuAsnLeuAspLysLys                              445450455                                                                     GCAGAGCTGTTAGAAGCGAAAGATTCTAATGGTTTATCTGGATTGTTT1443                          AlaGluLeuLeuGluAlaLysAspSerAsnGlyLeuSerGlyLeuPhe                              460465470                                                                     GTAGCCTTGCATAATGGATGTGTAGAAACAATTATTGCTTATGGGAAA1491                          ValAlaLeuHisAsnGlyCysValGluThrIleIleAlaTyrGlyLys                              475480485                                                                     ATACTTCACACTGCAGACCTTACTCCACATCAGGCATCAAAATTACTG1539                          IleLeuHisThrAlaAspLeuThrProHisGlnAlaSerLysLeuLeu                              490495500                                                                     GCAGCAGAAGGCCCAAATGGGGTATCTGGATTAATTATAGCTTTTCAA1587                          AlaAlaGluGlyProAsnGlyValSerGlyLeuIleIleAlaPheGln                              505510515520                                                                  AATAGGAATTTTGAGGCAATAAAAACTTATATGAAAATAATAAAAAAT1635                          AsnArgAsnPheGluAlaIleLysThrTyrMetLysIleIleLysAsn                              525530535                                                                     GAAAATATTACACCTGAAGAAATAGCAGAACACTTGGACAAAAAAAAT1683                          GluAsnIleThrProGluGluIleAlaGluHisLeuAspLysLysAsn                              540545550                                                                     GGAAGTGATTTTCTAGAAATTATGAAGAATATAAAAAGC1722                                   GlySerAspPheLeuGluIleMetLysAsnIleLysSer                                       555560565                                                                     (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal fragment                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       AlaProProVal4                                                                 (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal fragment                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       AlaProProValProIleAsnProAlaXaaProIleXaaArg14                                  (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: synthetic DNA                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       TAATCGAATTCATGGAATCCCTGACGTTA29                                               (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: synthetic DNA                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GGTACCCCCAATATTAGGGCCATCAACGTCAACGTTGCCGCC42                                  (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: synthetic DNA                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       AATATTGGGGGTACCGGTACTTATTTGGTCGAAGGCGATGCA42                                  (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: synthetic DNA                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       TGATAAGTCGACTCAGGCTGCCTGGCTAAT30                                              (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: synthetic DNA                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GGGGAATTCCAAATTCACAAATTTTTTTGT30                                              (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: synthetic DNA                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      TCCATGCCATTCATGGAGTATTAATGAATT30                                              (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: synthetic DNA                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      CTCCATGAATGGCATGGAAAGGCGGAATA29                                               (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: synthetic DNA                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      CGGGTCGACTCAGAAGGTATATTTCACACCCAA33                                           (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: synthetic DNA                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      CAGTGTATCACCACGAG17                                                           (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: synthetic DNA                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      AAATTATCTACAGTCAG17                                                           (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 723 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: genomic DNA                                               (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Shigella flexneri 2a                                            (B) STRAIN: M4243                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      ATGGTTCAGCGTAATATTCCCTTCATACTGGCTCCTGTCATTCACGGT48                            MetValGlnArgAsnIleProPheIleLeuAlaProValIleHisGly                              151015                                                                        GTCCGGGACAGAGGTACCTTCCTCCGGAATGACATAATTTCCTGTTCC96                            ValArgAspArgGlyThrPheLeuArgAsnAspIleIleSerCysSer                              202530                                                                        GTCATTTTTATCCACAAATGCCCTGTCACTTCCCAGTGTGATATGGCT144                           ValIlePheIleHisLysCysProValThrSerGlnCysAspMetAla                              354045                                                                        GTTATCCGACTTAATGTCACTGTTCAGCGAGGCGTTACGTGAAAGATG192                           ValIleArgLeuAsnValThrValGlnArgGlyValThr*LysMet                                505560                                                                        GAAGTCAGCGTCTTTCAGCGACAGTGTTTTCATTGTAAACTGACGGTT240                           GluValSerValPheGlnArgGlnCysPheHisCysLysLeuThrVal                              65707580                                                                      TTCCCAGTCTTTCTGGTTCAGGCTGACCGGTGCACTGCCACTGATGGA288                           PheProValPheLeuValGlnAlaAspArgCysThrAlaThrAspGly                              859095                                                                        GGCATGGATAACCGGATGTCCCTGGAATATCAGGGTGCCACTGTCCTG336                           GlyMetAspAsnArgMetSerLeuGluTyrGlnGlyAlaThrValLeu                              100105110                                                                     ACTCAGGGTACCTTCCGGCAGGTTCACGCTACCATCAAAGATTACCTT384                           ThrGlnGlyThrPheArgGlnValHisAlaThrIleLysAspTryLeu                              115120125                                                                     TCTTCCCCCCGGCACCTGTGGAATGGCGACATCCATATTCCCGGTCAG432                           SerSerProArgHisLeuTrpAsnGlyAspIleHisIleProGlyGln                              130135140                                                                     CTGACCATGAAAGATAACGGGTTGTTTTGCCCGCCCGGCCAGGATCCT480                           LeuThrMetLysAspAsnGlyLeuPheCysProProGlyGlnAspPro                              145150155160                                                                  ATCTTTTACTGTCTGAACTGCTTTGTTTTTGTTCATGCCAACAAACTC528                           IlePheTyrCysLeuAsnCysValValPheValHisAlaAsnLysLeu                              165170175                                                                     CCACTGAGCCGGATCATTCAGGCTGTTCCCCCACAGAGTGTTACCATA576                           ProLeuSerArgIleIleGlnAlaValProProGlnSerValThrIle                              180185190                                                                     GCTGGCAGATTTCAGAATATAGAAGCGGGTCTGGCTGTTGAGTATCAT624                           AlaGlyArgPheGlnAsnIleGluAlaGlyLeuAlaValGluTyrHis                              195200205                                                                     GCTGTACAGGTTTCCTGGAGTGCCGGTACCACCAAAGGGGGATATATT672                           AlaValGlnValSerTrpSerAlaGlyThrThrLysGlyGlyTyrIle                              210215220                                                                     TCCAATCGTCGGTTCACTGACATTTGTATCCTGAGCCTTAAGATCCAG720                           SerAsnArgArgPheThrAspIleCysIleLeuSerLeuLysIleGln                              225230235240                                                                  TAA723                                                                        __________________________________________________________________________

What is claimed is:
 1. An isolated DNA molecule encoding ShET1 whichconsists of the amino acid sequence encoded by the DNA of SEQ ID NO:15.2. The isolated DNA molecule of claim 1, wherein said DNA moleculeconsists of the nucleotide sequence shown in SEQ ID NO:15.
 3. A mutantShigella flexneri 2a which fails to produce any enterotoxic ShET1, ShET2or both, as a result of a mutation in the ShET1, ShET2 or both genes. 4.The mutant Shigella flexneri 2a of claim 3, wherein said mutation is adeletion mutation.
 5. The mutant Shigella flexneri 2a of claim 4,wherein said mutant has an aro⁻ and VirG⁻ phenotype.
 6. The mutantShigella flexneri 2a of claim 3, wherein said mutation is introducedinto parent strain Shigella flexneri 2a strain CVD1203 (ATCC NO. 55556).7. A plasmid comprising the DNA of claim
 1. 8. A plasmid comprising theDNA of claim 2.